Occurrence rate and species and subtypes of Cryptosporidium spp. in pet dogs in Yunnan Province, China

Background Cryptosporidium spp. is a ubiquitous, globally distributed intestinal protozoan infecting humans and at least 260 animal hosts. Due to close human contact with pet dogs and identification of zoonotic Cryptosporidium species and subtypes in these animals, dog health is not only a veterinarian issue but also a public health issue. This study aimed to understand occurrence and genetic characterization at both genotype and subtype levels in pet dogs in Yunnan Province, China. Results A total of 589 fresh fecal specimens were collected from adult pet dogs in the rural areas of eight cities/autonomous prefectures of Yunnan Province, China. 16 fecal specimens were positive for Cryptosporidium spp. by polymerase chain reaction (PCR) amplification and sequence analysis of the small subunit ribosomal RNA (SSU rRNA) gene, with an average occurrence rate of 2.7% (16/589) being observed. Three zoonotic Cryptosporidium species were identified: C. parvum (n = 7), C. suis (n = 5) and C. canis (n = 4). At the 60-kDa glycoprotein (gp60) locus, only three C. parvum and two C. canis specimens were successfully amplified and sequenced, with subtype IIaA17G2R1 (n = 3) and subtypes XXa4 (n = 1) and XXa5 (n = 1) being identified, respectively. Conclusions The present finding of three zoonotic Cryptosporidium species in dogs implied that dogs infected with Cryptosporidium spp. may pose a threat to human health. C. suis was identified in dogs in this study for the first time, expanding the host range of this species. Identification of C. parvum subtype IIaA17G2R1 and C. canis subtypes XXa4 and XXa5 will be helpful to explore the source attribution of infection/contamination and assess the transmission dynamics of C. parvum and C. canis in the investigated areas in the future.


Background
Pets are widely recognized as an integral part of modern society worldwide.Pet dogs are the most common companion animals, with the number of pet dogs increasing in recent years.The population of pet dogs was estimated to be between 83.7 million and 88.9 million in 2020, up 9.0-16.0%from year-end 2016 (https:// avmaj ourna ls.avma.org/ displ ay/ post/ news/ pet-popul ation-still-onthe-rise--with-fewer-pets-per-house hold.xml).However, dogs have been reported to be reservoirs, carriers, and transmitters of some parasites, including zoonotic intestinal protozoan Cryptosporidium spp.[1].Since the first report of this parasite in dogs in 1981, more than 100 publications have documented Cryptosporidium infections in dogs from 27 different countries worldwide [2,3].
Cryptosporidium spp. is a ubiquitous, globally distributed intestinal protozoan infecting humans and animals.Infectious Cryptosporidium oocysts are excreted with host feces into environments.Humans can acquire Cryptosporidium infections through the fecal-oral route, and four modes of transmission have been demonstrated: person-to-person transmission, animal-to-person transmission, foodborne transmission, and waterborne transmission [4].Due to the wide animal host range of Cryptosporidium spp.(at least 260 species), the epidemiological role animals play in the transmission of human cryptosporidiosis has been receiving more concern [5].Many studies have indicated that contact with animals is considered a risk factor for human Cryptosporidium infection [6].In fact, to date, more than 20 outbreaks of cryptosporidiosis related to contact with animals have been reported [7].Recent two-decade molecular epidemiological studies documented occurrence (average rate: 5.3% (929/17409)) of Cryptosporidium spp. in dogs, and zoonotic C. canis and C. parvum are the two most common species in these animals (Table 1).The findings above have raised awareness of zoonotic transmission potential of cryptosporidiosis due to contact with dogs.
Within the genus Cryptosporidium, extensive genetic variation has been confirmed.Currently, PCR-based molecular techniques targeting the small subunit of ribosomal RNA (SSU rRNA) gene have been widely used in the identification of Cryptosporidium species/genotypes.To date, at least 49 valid Cryptosporidium species and more than 120 genotypes have been described, with 23 species and two genotypes being identified in both humans and animals [60][61][62] 1).Since more than 100 confirmed human C. canis infections have been reported, the public health problem that dogs cause as a reservoir host of Cryptosporidium spp.should be paid more attention [63].A recently developed gp60 subtyping tool for C. canis has greatly improved our understanding of the transmission of this species [63].The observation of three C. canis subtypes (XXa1, XXa4 and XXb2) identified in both dogs and humans supports the potential of zoonotic transmission of C. canis from dogs to humans [25,63].
In  1).In southern China, especially in rural areas where ethnic minorities congregate, keeping dogs nearly becomes a common habit of inhabitants under the influence of local customs and cultures.The dogs generally serve well as protectors and companions.The aim of this study was to determine occurrence rate of Cryptosporidium spp.and distribution of species and genotypes/subtypes in pet dogs from the rural areas of eight cities/autonomous prefectures of Yunnan Province, China.Meanwhile, the potential of zoonotic transmission and the significance of public health of dog-derived Cryptosporidium isolates were also assessed by analyzing their genetic characterization at both genotype and subtype levels.

Occurrence rates of Cryptosporidium spp. in pet dogs
A total of 16 (2.7%) of 589 fecal specimens were determined to be infected with Cryptosporidium spp.by PCR amplification and sequence analysis of the SSU rRNA gene.Cryptosporidium spp. was found in pet dogs in six autonomous prefectures/cities.The occurrence rates were 6.0% in Zhaotong, 3.7% in Kunming, 3.4% in Dali Bai, 3.4% in Nujiang Lisu, 2.0% in Lijiang and 0.7% in Diqing Tibetan.There was an absence of Cryptosporidium spp. in pet dogs in Chuxiong Yi Autonomous Prefecture and Baoshan City of Yunnan Province (Table 2).

Molecular characteristics of
number: MT954607) and two subtypes of C. canis were identified: XXa4 and XXa5.

Discussion
Dogs have been reported to be infected naturally with zoonotic Cryptosporidium spp.Thus, the detection of Cryptosporidium spp. in dogs is not only a veterinarian issue but also a public health issue.In this study, the occurrence rate of Cryptosporidium spp. was 2.7% in the investigated dogs, which was lower than the pooled rate in dogs worldwide (5.3%) (Table 1).In fact, the occurrence rates of Cryptosporidium spp.are complicated in animals and probably affected by many factors, including age and health status of animals and their living conditions, and so on.Cryptosporidium spp. is considered as a causative agent of opportunistic infection.Health status of hosts is the key factor for its infection, and it is closely associated with host ages.Currently, a lot of studies on the relationship between the occurrence rates of Cryptosporidium spp.and the age of dogs indicated that Cryptosporidium infections are the most common in puppies (< 1-year-old dogs) [8,20,28,52,59,64,65].This finding may be related to the immature immune system of young animals.In a meta-analysis of the global prevalence of Cryptosporidium infection in dogs, a positive relationship was also observed between Cryptosporidium infection and diarrhea in dogs [3].A recent study demonstrated that the risk of being positive for Cryptosporidium spp. was 2.9-fold higher in the diarrheal dogs than in the no-obvious-clinical-sign dogs [55].The occurrence rate of Cryptosporidium spp. was significantly higher in diarrheal dogs than in non-diarrheal ones in two studies: 30.0% vs. 4.2% in Northeast Spain [66]; 30.3% vs. 16.9% in Nigeria [67].On the contrary, a higher but non-significant prevalence of Cryptosporidium spp. was found in non-diarrheic dogs than in diarrheic dogs in Thailand (32.6% vs. 23.5%)[58].The inconsistent conclusion may be related to the number of infecting Cryptosporidium oocysts.Additionally, living conditions of dogs might have influence on Cryptosporidium infection [68].Based on current dog epidemiological data of Cryptosporidium spp., it was observed that dogs in pet markets had the highest pooled occurrence rate (13.6%, 213/1570) (Table 1).Usually, the dogs in pet markets have the limited living spaces, leading to close contact with each other.Meanwhile, insufficient sanitary control of facilities easily induces transmission of Cryptosporidium spp.. Recently, there were two studies revealing that outdoor dogs had a higher prevalence of Cryptosporidium infection than indoor dogs conducted in Nigeria (26.1% vs. 14.7%) and Iran (79.4% vs. 20.6%)[49,69].In fact, early in 2007, Rambozzi et al., reported that outdoor dogs were approximately five times more likely to be infected with Cryptosporidium spp.than indoor ones [70].This phenomenon can be attributed to the fact that unconfined/ non-sheltered dogs can roam about in the streets for hunting, foraging and eating adulterated food materials.This increases the risk of getting infected with Cryptosporidium spp.In this study, all the dogs were adult and had no clinical signs of illness at the time of sampling.Meanwhile, all the dogs are from rural areas, and often kept in the courtyard.The limited range of dog activities might be a relatively low risk of Cryptosporidium infection for these animals.
In this study, sequence analysis of Cryptosporidium spp. at the SSU rRNA locus identified three zoonotic Cryptosporidium species (C.parvum, C. canis and C. suis), suggesting the potential of dog-related zoonotic transmission of Cryptosporidium spp. in the investigated areas.
C. parvum is often found in dogs worldwide, and sometimes showed predominance in these animals.In a recent study of molecular detection of Cryptosporidium spp. in dog fecal specimens contaminating public areas in Northern Italy, C. parvum accounted for > 90.0% of Cryptosporidium-positive specimens [57].In fact, C. parvum is one of the two most common Cryptosporidium species causing human cryptosporidiosis [71].Epidemiological studies have identified C. parvum in more than 155 mammalian species [5].Thus, C. parvum is of concern as a major zoonotic Cryptosporidium species.In this study, C. parvum subtype IIaA17G2R1 was identified in dogs for the first time.In fact, molecular epidemiological studies of Cryptosporidium spp., revealed that subtype IIaA17G2R1 was relatively common in livestock, occurring mainly in cattle [72] but appearing occasionally in other animals including sheep, camels, goats, rodents, mustangs and fish [73][74][75][76][77][78].This subtype has also been reported in human cases of cryptosporidiosis in Iran and Jordan [79,80].In North Carolina of the US in 2009, there was an outbreak of human cryptosporidiosis caused by subtype IIaA17G2R1 in a youth summer camp.Meanwhile, this subtype was identified in both humans and livestock in the camp setting, indicating that zoonotic transmission had occurred [81].Our finding of subtype IIaA17G2R1 in dogs suggests necessity to take measures to reduce the risk of zoonotic transmission of Cryptosporidium spp.from dogs to humans.
C. canis is the most often identified species in dogs and is considered a host-adapted species in dogs [68].The global epidemiologic data revealed that 86.8% (585/674) of dog-derived Cryptosporidium isolates belonged to C. canis (Table 1).C. canis was also found in some other animals, including foxes, coyotes, minks, mongooses, raccoon dogs, cattle and sheep as well as rodent animals [82][83][84].C. canis is one of common zoonotic Cryptosporidium species.However, most human infections with C. canis were reported in people in developing countries or those who have traveled to developing countries [63].C. canis was responsible for as much as 4.0% of overall cryptosporidiosis cases in children in developing countries [85].With the increasing case number of human cryptosporidiosis caused by C. canis (more than 100 cases worldwide), the transmission characteristics and public health significance of C. canis need to be studied extensively and deeply [63].A recently developed gp60 subtyping tool for C. canis can improve our understanding of the transmission of this zoonotic species.In this study, C. canis isolates were further subtyped, and a known subtype XXa4 and a novel subtype XXa5 were identified.Subtype XXa4 of C. canis has been reported in dogs, foxes and raccoon dogs and humans from Ethiopia and Peru (Table 3).To date, according to gp60 subtyping data of C. canis, a total of 20 subtypes belonging to nine subtype families (XXa-XXi) have been identified including subtype XXa5 newly identified in this study [50], and 11 subtypes (XXa1, XXa4, XXa5, XXb1-XXb3, XXd1, XXe1, XXe2, XXh1 and XXi1) could be seen in dogs (Table 3).Six subtypes (XXa1-XXa4, XXb2 and XXc1) have been reported in humans, with four subtypes being identified in both humans and animals: XXa1, XXa4 and XXb2 in dogs, XXa2 and XXa4 in raccoon dogs and XXa4 in foxes (Table 3), indicating the zoonotic potential of these subtypes.In fact, the occurrence of zoonotic transmission of cryptosporidiosis caused by subtype XXa4 has been confirmed based on the fact that the children and their dogs in a household belonged to subtype XXa4 [63,86].
To our knowledge, C. suis was reported in dogs for the first time in this study.It is well-known that C. suis is the dominant species infecting pigs (especially in pre-weaned pigs) [89].The accidental finding of C. suis in dogs might be closely related to the fact that coprophagia is a frequent practice in these animals.In the investigated areas, almost all the families raised pigs for consumption.Combined with the observation of C. suis mostly found in pigs previously [90], pigs were suspected to be the source of C. suis infection in dogs in the investigated areas.Dogs could acquire C. suis infections by exposure to fecal materials from pigs in their environment.C. suis was also found in other animals, such as cattle, red deer and rodent animals [91][92][93].Human cases of cryptosporidiosis caused by C. suis are occasionally reported in some countries, including China, Thailand, Cambodia, Peru, the UK and Madagascar [91,[94][95][96][97][98].Currently, we could not confirm actual infections or mechanical transmission of C. suis in dogs only based on molecular detection of C. suis in dog fecal specimens.The epidemiological role of dogs in the transmission of cryptosporidiosis caused by C. suis needed to be assessed.

Conclusions
In conclusion, this study described the occurrence (2.7%, 16/589) and genetic characterization of Cryptosporidium spp. at both genotype and subtype levels in pet dogs in Yunnan Province, China.The finding of three zoonotic Cryptosporidium species (C.parvum, C. canis and C. suis) in dogs implied that dogs infected with Cryptosporidium spp.may pose a threat to human health.High frequency of human contact with pet dogs increases the

Fecal specimen collection
During April to June 2021, 589 fecal specimens (each approximately 15-20 g) were collected from adult pet dogs from the rural areas of eight cities/autonomous prefectures of Yunnan Province, China.Detailed sampling sites and specimen numbers were shown in Table 2.One fecal specimen of each animal was taken immediately from fresh feces on the ground after defecation using a sterile disposable latex glove and was then placed into plastic zippered bags individually.Meanwhile, each specimen was given a unique identification number.No clinical signs of diarrhea were observed in any of the animals at the time of specimen collection.All the collected specimens were transported to our laboratory in a cooler with ice packs and were stored at − 20 °C in a freezer.

DNA extraction
Genomic DNA was extracted from approximately 180-200 mg of each specimen using a QIAamp DNA Stool Mini Kit (QIAgen, Hilden, Germany), and the procedures and reagents utilized were provided by the manufacturer.The lysis temperature was increased to 95 °C due to difficulty in lysing Cryptosporidium oocysts.The extracted DNA (200 µl) was stored at − 20 °C until being analyzed by PCR.

Cryptosporidium genotyping and subtyping
The partial small subunit (SSU) rRNA gene (approximately 830 bp) was amplified by nested PCR to identify Cryptosporidium species and genotypes.The primers and the cycling parameters were used as described previously by Huang et al. [99].The C. parvum-and C. canis-positive specimens were analyzed to determine subtypes by nested PCR amplification of the gp60 gene (approximately 400 bp for C. parvum and 700 bp for C. canis).The primers and the cycling parameters were used as described previously by Sulaiman et al. for C. parvum [100] and by Jiang et al. for C. canis [63].Two replicates were used in PCR analysis of each specimen at each locus to increase detection rate, with both negative and positive controls being included in each PCR test.All secondary PCR products were subjected to electrophoresis in 1.5% agarose gels and visualized by staining the gel with GelStrain (TransGen Biotech., Beijing, China).

Nucleotide sequencing and analyzing
For each target gene, all positive secondary PCR products of expected size were sequenced in both directions on an ABI PRISM 3730XL DNA Analyzer by Comate Bioscience Company Limited (Jilin, China), using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Carlsbad, CA, USA).All raw sequencing data obtained in the present study were assembled using the software MEGA 11.0 (https:// www.megas ofware.net/).Edited nucleotide sequences were subjected to BLAST searches (http:// www.ncbi.nlm.nih.gov/ blast/) to determine Cryptosporidium species and subtypes.

Table 1
Prevalence of Cryptosporidium spp.and distribution of species and genotypes/subtypes in dogs worldwide (1); C. hominis(1); C. ubiquitum(1); C. andersoni(1); C. scrofarum(1); C. proliferans(1)polymorphic sites being observed.Five different C. suis sequences (accession number: PP897004-PP897008) were found with nine polymorphic sites being observed.In contrast, four obtained C. canis sequences were identical to each other (accession number: PP896998).Molecular characteristics of Cryptosporidium spp. at the gp60 LocusSeven C. parvum and four C. canis DNA specimens were further subtyped by PCR amplification and sequence analysis of the gp60 gene.However, only three C. parvum and two C. canis specimens produced the expected PCR amplicons and were successfully sequenced.Three gp60 gene sequences of C. parvum were identical to each other (accession number: PP887783) and had 100% similarity with the sequence of subtype IIaA17G2R1 (accession number: PP112337).Two gp60 gene sequences of C. canis (accession number: PP887781-PP887782) had two base differences.They had the largest similarity (100% and 99.7%) with the sequence of subtype XXa4 (accession

Table 1 (
continued) (17)iferans(1); C. canis+C.parvum(1);C.spp.(17)Note:C. spp.indicated that Cryptosporidium spp.were untyped a The prevalences of Cryptosporidium spp.were determined by morphological or immunological methods.Only some of Cryptosporidium-positive specimens were successfully genotyped by PCR and sequencing b Of the 11 infected dogs, only specimens from three dogs living with infected children were subjected to molecular analysis c Others contained the dogs not having the specific number of specimens related to the sources or having unclear sources d Only Cryptosporidium-positive specimens were genotyped in the study conducted in the Czech Republic

Table 2
Occurrence of Cryptosporidium species and subtypes in pet dogs in Yunnan Province, China a N and E indicate north latitude and east longitude, respectivelySampling site (N, E) aExamined no.Positive no.(%) Species (n) Subtypes (n)

Table 3
Host and geographical distribution of gp60 subtypes of Cryptosporidium canisNote: Bold letters indicate the newly discovered subtype in this study risk of human Cryptosporidium infection.Therefore, health education to reduce dog-related zoonotic transmission of Cryptosporidium infection should be carried out among people, especially pet owners and veterinarians having close contact with these animals.C. suis was identified in dogs in this study for the first time, expanding the host range of this species.Identification of C. parvum subtype IIaA17G2R1 and C. canis subtypes XXa4 and XXa5 will be helpful to explore the source attribution of infection/contamination and assess the transmission dynamics of C. parvum and C. canis in the investigated areas in the future.